JPH04288083A - New antibiotic MBA 028-24A substance and its manufacturing method - Google Patents

Abstract

PURPOSE:To obtain a new antibiotic substance capable of exhibiting powerful growth inhibitory activity against Gram-positive bacteria and molds. CONSTITUTION:An actinomyces Streptomyces.hygroscopicus A1491 strain (FERM P-11864) belonging to the genus Streptomyces is cultured to produce a new antibiotic substance MBA 028-24A useful as an antimicrobial agent in the field of medicines, agricultural chemicals, etc.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides novel antibiotic MBA
The present invention relates to a substance MBA 028-24A and a method for producing the same, and more particularly to a novel antibiotic MBA 028-24A substance useful as an antibacterial agent in fields such as medicine and agrochemicals, and a method for producing the same.

0002

[Prior Art and Problems to be Solved by the Invention] Conventionally, many antibiotics have been known as physiologically active substances produced by microorganisms, and have been put to practical use in the fields of pharmaceuticals, veterinary drugs, agricultural chemicals, and the like. However, on the other hand, problems such as the appearance of resistant bacteria have arisen, and the current situation is that new antibiotics are constantly being sought.

0003

[Means for Solving the Problem] As a result of searching for a substance with excellent antibacterial activity, the present inventors discovered that such a substance was produced in a culture of a certain actinomycete belonging to the genus Streptomyces. They discovered this and completed the present invention.

That is, the gist of the present invention resides in a novel antibiotic MBA 028-24A substance having the following physicochemical properties and a method for producing the same. (a) Properties of substance: Colorless powder (b) Specific rotation: [α]25D = +60° (c
= 1.0, methanol) (c) Melting point: Gradually discolors and decomposes from 130°C (d
) Molecular weight: 1082.6708 (M+H)+FAB
(e) Ultraviolet absorption spectrum by -MS: λMAX nm (E1%1cm): 240.5 (3
78), 264.0 (199) (f) Infrared absorption spectrum: The infrared absorption spectrum measured by the liquid film method shows the following main absorptions: 3500-3200, 2960, 2940, 1700,
1640, 1590, 1460, 1380, 1240,
1140, 1090, 1070, 1000, 960 (
νcm-1)(g)1H-Nuclear Magnetic Resonance Spectrum:
Measurement was performed in heavy methanol using tetramethylsilane as an internal standard. The following major chemical shifts are shown (500
MHz) 0.69 (3H, d, J=7.0Hz), 0.
87 (3H, d, J = 7.0Hz), 0.91 (3H,
d, J=6.8Hz), 0.95 (3H, d, J=6.
8Hz), 1.01 (3H, d, J=6.7Hz), 1
．． 08 (3H, d, J=6.8Hz), 1.12-1.
55 (m), 1.57 (3H, s), 1.55-1.
87 (m), 1.91 (1H, m), 1.98 (2H
, m), 2.06 (2H, m), 2.38 (1H, m)
, 2.47 (1H, m), 2.84 (3H, s), 3.
15 (2H, t, J=7.1Hz), 3.77-3.9
4 (7-8H, m), 4.07 (1H, m), 4.2
5 (1H, m), 4.73 (1H, dd, J=9.0,
2.8Hz), 5.32 (1H, dd, J=14.8
, 8.8Hz), 5.43 (2H, m), 5.81 (1
H, d, J = 15.3Hz), 5.86 (1H, d, J
=10.7Hz), 6.17(1H, dd, J=14
．． 9,10.9Hz), 6.19~6.23(2H,
m), 7.18 (1H, m) (h) 13C-Nuclear Magnetic Resonance Spectrum: Measured in heavy methanol using tetramethylsilane as an internal standard. Showing the following major chemical shifts (500MHz)9.
9, 10.3, 11.6, 12.3, 13.5, 13
．． 7, 16.8, 27.9, 28.3, 29.7, 30
．． 5, 31.5, 33.6, 34.0, 34.4, 34
．． 7, 38.3, 41.0, 41.1, 41.3, 41
．． 8, 42.0, 42.1, 43.6, 43.9, 45
．． 1, 45.2, 64.8, 65.3, 67.2, 69
．． 6, 70.0, 71.7, 72.7, 76.0, 76
．． 4, 79.9, 80.7, 99.5, 120.5,
128.1, 128.8, 130.1, 132.3
, 136.5 , 138.4 , 146.8 , 14
8.8, 158.1, 168.9, 171.6
, 174.0 (i) Solubility: Soluble in methanol, aqueous acetone, aqueous tetrahydrofuran Slightly soluble or insoluble in water, acetone, tetrahydrofuran, acetonitrile, ethanol (j) Color reaction: Dragendorff reagent, phosphomolybdic acid, Positive to 10% sulfuric acid Negative to ninhydrin, Ehrlich's reagent, ferric chloride The present invention will be described in detail below. The novel antibiotic MBA 028-24A substance of the present invention (hereinafter referred to as “MBA
028-24A substance) can be obtained, for example, from a culture solution of a MBA 028-24A substance-producing bacterium that belongs to the genus Streptomyces and has the characteristics and properties shown below. Such producing bacteria include Streptomyces hygroscopicus.
s hygroscopicus) A1491 (hereinafter sometimes abbreviated as "this strain" or "A1491 strain"). This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with the number 11864 (FERM).
P-11864).

[0005] The morphological characteristics, properties on various media, and physiological/biological properties of this strain are as shown below. a) Morphological characteristics Starch/inorganic salt agar medium and glycerin/asparagine agar medium were used as the sporulation medium at 27°C for 1
Observation was made after culturing for 4 days. Colony color is white to gray;
As time passes, the aerial mycelia become moist and darken. The basal hyphae branch and elongate, and no division is observed. Most of the spore chains have a dense spiral shape, and the spore segments are not clearly defined. The surface structure is warty ~
Shows a rough surface.

b) Properties on each medium ISP [International Streptomyces Project (I)
international streptomyce
s Project)] at 27°C on various specified media.
The properties after 14 days of culture are as shown below. below,
G indicates growth, RC indicates color tone of the back surface, AM indicates aerial hyphae, SP indicates soluble pigment, and S indicates properties of the A1491 strain regarding spore formation.

1) Sucrose-nitrate agar medium G:
Slightly poor, white to gray RC: Gray AM: Slightly weak, powdery, white SP: Not produced S: Vigorous, dense spiral 2) Glucose-asparagine agar medium G: Moderate,
Yellowish white RC: Light yellow AM: Slightly poor, powdery, white SP: Not produced S: Poor, dense spiral 3) Glycerin-asparagine agar medium (ISP5) G
: Medium, white to gray, partially moistened RC: Light yellow AM: Abundant, velvety to powdery SP: No formation S: Medium, dense spiral 4) Starch/Inorganic Salt Agar Medium (ISP4) G: Good, gray to dark gray RC: Light yellow AM: Moderate SP: Not produced S: Vigorous, dense spiral 5) Tyrosine agar medium (modified method by Okanishi) G: Moderate RC: Light yellow AM: Abundant, powdery, white SP: Not formed S: Not formed 6) Ordinary agar medium G: Medium, gray to light olive gray RC: Light yellowish brown AM: Abundant, powdery, gray SP: Not formed S : Not formed 7) Yeast extract/malt extract agar medium (ISP2) G:
Slightly poor, gray to black, mostly moist RC: Light yellowish brown AM: Abundant, powdery, gray SP: Not produced S: Fairly poor 8) Oatmeal agar medium (ISP8) G: Moderate;
White to light brown-gray RC: Light yellow AM: Medium, velvety, white SP: No formation S: Vigorous, dense spiral 9) Bennett Agar G: Good, light olive gray to black, partially moistened R.C.
: Light yellowish brown AM: Abundant, velvety to powdery, bright olive gray SP: Not produced S: Vigorous, dense spiral 10) Calcium malate agar medium G: Poor, yellowish white RC: Yellowish white AM : Extremely poor SP: Not produced S: Not formed and has no ability to assimilate calcium malate C) Physiological properties Physiological properties of the A1491 strain 1 to 3 weeks after culture are shown. Hereinafter, + means positive, - means negative, and ± means doubtful.

1) Litmus milk peptonization: + Coagulation: - 2) Reduction of nitrate: - 3) Production of melanin-like pigments PYFe medium: - Try-Y medium: - Tyrosine medium: - 4) Hydrolysis of starch: + 5) NaCl tolerance 4%: + 7%: - 10%: - 6) Production of acids from various sugars D-glucose: + L-arabinose: + D-xylose: + D-fructose: + Sucrose: + L -Rhamnose: ± Raffinose: + Inositol: + 7) Growth temperature range: 15 to 45°C Note) PTFe medium: Peptone, yeast extract, iron agar medium Try-Y medium: Tryptone, yeast extract agar medium d) Regarding bacterial components The cell wall amino acid type of the present strain A1491 was confirmed to be cell wall type I, since L,L-diaminopimelic acid was detected by analysis of whole cell hydrolyzate. Regarding the cell wall sugar composition, glucose and ribose were detected in the whole cell hydrolyzate, but no characteristic pattern was observed.

e) Taxonomic considerations The colony color of this strain A1491 is white to grayish-brown at the early stage of culture, and as the culture gets older, black spots appear here and there on the colony, which gradually spreads and forms the entire colony. becomes black and wet. Does not produce melanin pigment. The sporulation mode shows a dense spiral. The spore surface is warty to rough. Based on the above properties, the A1491 strain is Streptomyces
It is clear that it is an actinomycete belonging to the genus es.

[0010] Bacterial species with the properties shown by A1491 strain were
ergey's Manual 8th Edition, ISP (In
International Journal of
Systematic Bacteriology
, Vol. 22, pp. 307-311, 1972) and other literature, we found that Streptomyces hygroscopici is the most closely related species.
Cus was mentioned.

[0011] Streptomyces hygro
Scopicus 1) forms a helical spore chain at the tip of the aerial hyphae, and the spore surface is warty to rough; 2)
It has three major characteristics: it grows gray-brown aerial mycelium, and 3) the aerial mycelium becomes moist in the later stages of cultivation and becomes black-spotted.

[0012] As stated above, the A1491 strain is ISP
Streptomyceshygroscopy described
cus and Streptomyces
The strain was identified as St. hygroscopicus.
reptomyces hygroscopicus
It was named A1491.

[0013] The present invention is not limited to the A1491 strain, but any strain capable of producing the above-mentioned novel antibiotic MBA 028-24A substance can be used. In the present invention, the above-mentioned bacteria are cultured in a medium containing a nutrient source that can be used by ordinary microorganisms. This medium may be any medium containing nutritional sources that can be used by actinomycetes, and its components include sugars, starches, assimilable carbon sources such as glycerin, corn steep liquor, yeast extract, meat extract, peptone, and ammonium. Organic or inorganic nitrogen sources such as salts can be included. Moreover, phosphates, metal salts such as calcium, iron, etc. can also be contained. In addition, silicone as needed.
Vegetable oils or synthetic antifoaming agents may be added to the medium.

[0014] As a culture method, a culture method under aerobic conditions is suitable. The appropriate temperature for culturing is 10 to 30°C, but in many cases, culture is carried out at 20 to 27°C. M of the present invention
The production of BA028-24A substance varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 3 to 10 days in both shaking culture and tank culture.

[0015] When the amount accumulated in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution. The MBA 028-24A substance of the present invention can be collected from the culture solution using any commonly used means. for example,
Means utilizing the solubility difference between the MBA 028-24A substance and the impurity, means utilizing the difference in adsorption affinity, and means utilizing the difference in ionic binding force may be used alone or in combination, or repeatedly.

[0016]

EXAMPLES The present invention will be explained in more detail with reference to examples below, but the present invention is not limited to the following examples unless it exceeds the gist thereof.

(Example 1) Starch syrup 4.0%, soybean oil 0.3%, soybean flour 2.0%, Pharmamedia 1.0%, sungrain 0.5%, CaCO3 0
．． 3%, FeSO4 ・7H2 O 0.0001%
, CoCl2 ・6H2 O 0.00001%, N
80 ml of medium containing 0.00001% iCl2 (
Dispense the solution (pH 7.0) into a 500 ml Erlenmeyer flask, sterilize it, and add Streptomyces hygroscopicus A1491.
The strain was inoculated and cultured with shaking at 210 rpm for 96 hours at 27°C. 20 liters of medium with the same composition as above (pH
200 ml of the above culture solution was inoculated into a 30 liter jar fermenter containing 7.0), and cultured with aeration (20 liter/min aeration, 200 rpm) at 27° C. for 96 hours. Then, the culture solution was collected, and the bacterial cells were filtered out from the culture solution. 4 liters of acetone was added to the obtained bacterial cells, and the cells were thoroughly immersed, stirred, and the extract was filtered. Distilled water was added to this extract to adjust the volume to 7 liters, and this was saturated with 60% methanol beforehand.
The top of 20 columns (approximately 1 liter) was packed. After washing with 60% methanol, the active ingredient was eluted with methanol. The obtained active fractions were collected and the solvent was distilled off under reduced pressure to obtain 21 g of crude powder.

(Example 2) In order to further purify the crude powder of Example 1, 10 g of this crude powder was dissolved in 60% methanol, and ODS-silica gel (Yamamura Kagakusha Co., Ltd.) was eluted with a gradient from 60% methanol to 100% methanol. Manufactured by O
Reverse phase partition chromatography was performed using DS-AQ 120-S50) and active fractions were collected. The solvent of this active fraction was distilled off under reduced pressure to obtain 5 g of pale yellow crude powder. This powder was subjected to reverse phase partition high performance liquid chromatography using a methanol-water mixture (85:15) as a developing solvent.
MC-D-ODS-5 column (manufactured by Yamamura Kagaku Co., Ltd., 30×2
50 mm) to obtain fractions containing the MBA 028-24A substance, and the solvent of each of these fractions was distilled off under reduced pressure to obtain a powder of about 0.8 mm of the MBA 028-24A substance.
I got g.

The measurement results of the UV absorption spectrum, IR absorption spectrum, 1H-nuclear magnetic resonance spectrum, 13C-nuclear magnetic resonance spectrum, and mass spectrum of the MBA 028-24A substance were as follows.

■UV absorption spectrum: As shown in FIG. 1, it exhibits characteristic absorption at 240.5 nm and 264.0 nm.

■IR absorption spectrum: As shown in FIG. 2, the IR absorption spectrum measured by the liquid film method shows the following main absorptions.

[0022] νcm-1: 3500-3200, 29
60, 2940, 1700, 1640, 1590, 14
60, 1380, 1240, 1140, 1090, 10
70, 1000, 960 (1) 1H-Nuclear Magnetic Resonance Spectrum: The results of measurement in heavy methanol using tetramethylsilane as an internal standard are shown in FIG. The following major chemical shifts are shown (500
MHz)

0.69 (3H, d, J=7.0
Hz), 0.87 (3H, d, J=7.0Hz), 0.
91 (3H, d, J = 6.8Hz), 0.95 (3H,
d, J=6.8Hz), 1.01 (3H, d, J=6.
7Hz), 1.08 (3H, d, J=6.8Hz), 1
．． 12-1.55 (m), 1.57 (3H, s), 1
．． 55-1.87 (m), 1.91 (1H, m), 1
．． 98 (2H, m), 2.06 (2H, m), 2.38
(1H, m), 2.47 (1H, m), 2.84 (3H
, s), 3.15 (2H, t, J=7.1Hz), 3.
77-3.94 (7-8H, m), 4.07 (1H,
m), 4.25 (1H, m), 4.73 (1H, dd,
J=9.0, 2.8Hz), 5.32(1H, dd,
J=14.8, 8.8Hz), 5.43 (2H, m),
5.81 (1H, d, J = 15.3Hz), 5.86
(1H, d, J=10.7Hz), 6.17 (1H,
dd, J=14.9, 10.9Hz), 6.19~6
．． 23 (2H, m), 7.18 (1H, m) ■13C-
Nuclear magnetic resonance spectrum: The results of measurement in heavy methanol using tetramethylsilane as an internal standard are shown in FIG. Shows the following major chemical shifts (500MHz)

[
9.9, 10.3, 11.6, 12.3,
13.5, 13.7, 16.8, 27.9, 28.3,
29.7, 30.5, 31.5, 33.6, 34.0,
34.4, 34.7, 38.3, 41.0, 41.1,
41.3, 41.8, 42.0, 42.1, 43.6,
43.9, 45.1, 45.2, 64.8, 65.3,
67.2, 69.6, 70.0, 71.7, 72.7,
76.0, 76.4, 79.9, 80.7, 99.5,
120.5, 128.1, 128.8, 130.
1, 132.3, 136.5, 138.4, 14
6.8, 148.8, 158.1, 168.9
, 171.6, 174.0 ■Mass spectrum: High resolution FAB-MS (Fa
st Atom Bombardment Ma
When measured by SS Spectrometry, it was 1082.6708 (M+H)+.

[0025] When the MBA 028-24A substance was structurally analyzed, it was confirmed that it was a compound represented by the following formula.

[0026]

[Chemical formula 1]

(In the above formula, one of R1 and R2 is -C
OCH2COOH, and the other represents a hydrogen atom) (Example 3) According to the Japanese Society of Chemotherapy standard method, determine the minimum growth inhibitory concentration (mcg/ml) of the MBA 028-24A substance obtained in the example for each test bacterium. , which is shown below.

[0028] Bacillus subtilis (
Bacillus subtilis): 3.1 Sarcina lutea:
3.1 Staphylococcus aureus
209P: 1.56 (Staphylococcus aureus 209P) Bacteroides fragilis: 6.2 Escherichia coli (Escherichia coli)
Cori): >100Pseudomonas aeru
ginosa (Pseudomonas aeruginosa): ＞
100 Cryptococcus neoformans (
Cryptococcus neoformans): 1.56 Trichophyton mentagrophyt
es (Trichophyton mentagrophytes): 3. 1 Candida albicans: 3.1 Aspergillus fumi
gatus (Aspergillus fumigatus): 3.
1 Alternaria pori: 3.1 Fusarium oxysporum
(Fusarium oxysporum): 12.5 Botrytis cinerea: 0.78 Piricularia oryz
ae (Pyricularia oryzae): 0.78 Rhizoctonia solani (Rhizoctonia solani): 0.78

[0029]

Effects of the Invention The antibiotic obtained by the present invention exhibits a strong growth-inhibiting ability against Gram-positive bacteria and filamentous fungi, and has a high activity in medicines such as therapeutic agents for bacterial and fungal infections, plant disease control agents, and agricultural chemicals. Useful as an ingredient.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum (in methanol) of the MBA 028-24A material.

FIG. 2 is a drawing showing an infrared absorption spectrum of MBA 028-24A substance measured by a liquid film method.

FIG. 3 shows the 1 H-nuclear magnetic resonance spectrum (500 MHz, in heavy methanol) of the MBA 028-24A material.

FIG. 4 shows the 13C-nuclear magnetic resonance spectrum (500 MHz, in deuterated methanol) of the MBA 028-24A material.

Claims (2)

[Claims] Claim 1: A novel antibiotic MBA 028-24A substance having the following physicochemical properties. (a) Properties of substance: Colorless powder (b) Specific rotation: [α]25D = +60℃ (c
= 1.0, methanol) (c) Melting point: Gradually discolors and decomposes from 130°C (d
) Molecular weight: 1082.6708 (M+H)+FAB
(e) Ultraviolet absorption spectrum by -MS: λMAX nm (E1%1cm): 240.5 (3
78), 264.0 (199) (f) Infrared absorption spectrum: The infrared absorption spectrum measured by the liquid film method shows the following main absorptions: 3500-3200, 2960, 2940, 1700,
1640, 1590, 1460, 1380, 1240,
1140, 1090, 1070, 1000, 960 (
νcm-1)(g)1H-Nuclear Magnetic Resonance Spectrum:
Measurement was performed in heavy methanol using tetramethylsilane as an internal standard. The following major chemical shifts are shown (500
MHz) 0.69 (3H, d, J=7.0Hz), 0.
87 (3H, d, J = 7.0Hz), 0.91 (3H,
d, J=6.8Hz), 0.95 (3H, d, J=6.
8Hz), 1.01 (3H, d, J=6.7Hz), 1
．． 08 (3H, d, J=6.8Hz), 1.12-1.
55 (m), 1.57 (3H, s), 1.55-1.
87 (m), 1.91 (1H, m), 1.98 (2H
, m), 2.06 (2H, m), 2.38 (1H, m)
, 2.47 (1H, m), 2.84 (3H, s), 3.
15 (2H, t, J=7.1Hz), 3.77-3.9
4 (7-8H, m), 4.07 (1H, m), 4.2
5 (1H, m), 4.73 (1H, dd, J=9.0,
2.8Hz), 5.32 (1H, dd, J=14.8
, 8.8Hz), 5.43 (2H, m), 5.81 (1
H, d, J = 15.3Hz), 5.86 (1H, d, J
=10.7Hz), 6.17(1H, dd, J=14
．． 9,10.9Hz), 6.19~6.23(2H,
m), 7.18 (1H, m) (h) 13C-Nuclear Magnetic Resonance Spectrum: Measured in heavy methanol using tetramethylsilane as an internal standard. Showing the following major chemical shifts (500MHz)9.
9, 10.3, 11.6, 12.3, 13.5, 13
．． 7, 16.8, 27.9, 28.3, 29.7, 30
．． 5, 31.5, 33.6, 34.0, 34.4, 34
．． 7, 38.3, 41.0, 41.1, 41.3, 41
．． 8, 42.0, 42.1, 43.6, 43.9, 45
．． 1, 45.2, 64.8, 65.3, 67.2, 69
．． 6, 70.0, 71.7, 72.7, 76.0, 76
．． 4, 79.9, 80.7, 99.5, 120.5,
128.1, 128.8, 130.1, 132.3
, 136.5 , 138.4 , 146.8 , 14
8.8, 158.1, 168.9, 171.6
, 174.0 (i) Solubility: Soluble in methanol, aqueous acetone, aqueous tetrahydrofuran Slightly soluble or insoluble in water, acetone, tetrahydrofuran, acetonitrile, ethanol (j) Color reaction: Dragendorff reagent, phosphomolybdic acid, Positive to 10% sulfuric acid, negative to ninhydrin, Ehrlich's reagent, ferric chloride [Claim 2] The antibiotic MB according to Claim 1 is obtained by culturing actinomycetes belonging to the genus Streptomyces and using the cultured product.
A method for producing a novel antibiotic MBA 028-24A substance, which comprises collecting the A 028-24A substance.